The activation of proteins by post-translational modification represents an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. For example, protein phosphorylation plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes. In spite of the importance of protein modification, it is not yet well understood at the molecular level. The reasons for this lack of understanding are, first, that the cellular modification system is extraordinarily complex, and second, that the technology necessary to unravel its complexity has not yet been fully developed.
The complexity of protein modification, including phosphorylation, on a proteome-wide scale derives from three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging. The human genome encodes, for example, over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001). Each of these kinases phosphorylates specific serine, threonine, or tyrosine residues located within distinct amino acid sequences, or motifs, contained within different protein substrates. Most kinases phosphorylate many different proteins: it is estimated that one-third of all proteins encoded by the human genome are phosphorylated, and many are phosphorylated at multiple sites by different kinases. See Graves et al., Pharmacol. Ther. 82:111-21 (1999).
Many of these phosphorylation sites regulate critical biological processes and may prove to be important diagnostic or therapeutic targets for molecular medicine. For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases. See Hunter, supra. Oncogenic kinases such as ErbB2 and Jak3, widely expressed in breast tumors and various leukemias, respectively, transform cells to the oncogenic phenotype at least in part because of their ability to phosphorylate cellular proteins. Understanding which proteins are modified by these kinases will greatly expand our understanding of the molecular mechanisms underlying oncogenic transformation. Thus, the ability to identify modification sites, e.g. phosphorylation sites, on a wide variety of cellular proteins is crucially important to understanding the key signaling proteins and pathways implicated in disease progression, for example cancer.
The efficient identification of protein phosphorylation sites relevant to disease has been aided by the recent development of a powerful new class of antibodies, called motif-specific, context-independent antibodies, which are capable of specifically binding short, recurring signaling motifs comprising one or more modified (e.g. phosphorylated) amino acids in many different proteins in which the motif recurs. See U.S. Pat. No. 6,441,140, Comb et al. Many of these powerful new antibodies are now available commercially. See CELL SIGNALING TECHNOLOGY, INC. 2003-04 Catalogue. More recently, a powerful new method for employing such motif-specific antibodies in immunoaffinity techniques coupled with mass spectrometric analysis to rapidly identify modified peptides from complex biological mixtures has been described. See U.S. Patent Publication No. 20030044848, Rush et al.). Such techniques will enable the rapid elucidation of protein activation and phosphorylation events underlying diseases, like cancer, that are driven by disruptions in signal transduction.
One form of cancer, in which underlying signal transduction events are involve but still poorly understood, is Anaplastic Large-Cell Lymphoma (ALCL). ALCL is a sub-type of non-Hodgkin's lymphomas (NHL), which are the 5th most common cancer in the United States, with over 53,000 new diagnoses annually (source: The Leukemia & Lymphoma Society (2004)). Worldwide, more than 166,000 cases of NHL are diagnosed annually, and over 93,000 annual deaths from this group of lymphomas (source: Globocan 2000: Cancer Incidence, Mortality & Prevalence, Version 1.0 (2001)). ALCL, a form of T-cell lymphoma (CD30+), is most prevalent among young children, representing about 15% of all pediatric non-Hodgkin's lymphomas (source: UMDNJ Hematopathology (2004)). It is an aggressive disease that can be either systemic or primary cutaneous, with median survival rates of about 5 years from diagnosis.
Approximately 50% to 60% of all ALCL cases are characterized by a translocation between chromosomes 2p23 and 5q35 leading to an abnormal fusion gene involving the anaplastic lymphoma kinase (ALK) gene and the nucleophosmin gene (NPM), itself involved in nucleo-cytoplasmic trafficking. See, e.g. Ouyang et al., J. Biol. Chem. 278: 300028-300036 (2003); Miller, ProPath “Anaplastic Lymphoma Kinase” (2003). The ALK-NPM fusion protein functions as a constitutively activated protein tyrosine kinase, leading to enhanced cellular proliferation and survival. It has recently been shown that ALK-NPM transgenic mice spontaneously develop T-cell lymphomas including ALCL. See Chiarle et al., Blood 101: 1919-1927 (2003).
A number of downstream signaling protein targets of ALK-NPM have identified as potentially involved in mediating cellular transformation in ALK-NPM positive ALCL, including Shc, IRS-1, Grb2, phospholipase C-γ, P13-kinase, and Stat3/5. See Ouyang et al. supra; Zamo et al., Oncogene 21: 1038-1047 (2002). ALK-NPM activates the AKT/PI3K anti-apoptotic signaling pathway. Transgenic mice experiments have established that Stat3 and Jak3 are constitutively activated in ALK-NPM positive transgenic mice that develop ALCL. See Chiarle et al., supra. However, despite the identification of some of the downstream targets of ALK-NPM, the molecular mechanisms of contributing to ALK-NPM-mediated oncogenesis in ALCL remain incompletely understood. See Ouyang et al., supra.
A few phosphotyrosine sites that allow NPM-ALK to interact with other signaling proteins have been reported, including Tyr1604, which is a binding site for phospholipase gamma (PLCgamma) (see Bai et al. Mol. Cell. Biol. 18: 6951-6961 (1998), and Tyr1096 and Tyr1507, which are the docking sites for SHC and IRS-1 respectively. See Fujimoto et al., PNAS 93: 4181-4186 (1996). PLCgamma, SHC and IRS-1 are known to be phosphorylated in the context of other signaling cascades and many of their phosphorylation sites have been identified. See Watanabe et al., J. Biol. Chem. 276: 38595-38601 (2001); Law et al., Mol Cell Biol 16: 1305-1315 (1996); van der Geer et al., Curr. Biol. 6: 1432-1444 (1996); White M F, Mol. Cell. Biochem. 182: 3-11 (1998). Another important factor directly phosphorylated by NPM-ALK fusion kinase is STAT3. Phosphorylation of STAT3 at Tyr705 has been shown to be important for oncogenic transformation. See Zamo A. et al. Oncogene 21: 1038-1047 (2002).
Nonetheless, the small number of ALCL-related phosphorylation sites that have been identified to date do not facilitate a complete and accurate understanding of how protein activation within ALK-NPM signaling pathways is driving this disease.
Accordingly, there is a continuing need to unravel the molecular mechanisms of ALK-NPM driven oncogenesis in ALCL, by identifying the downstream signaling proteins mediating cellular transformation in this disease. Identifying particular phosphorylation sites on such signaling proteins and providing new reagents, such as phospho-specific antibodies and AQUA peptides, to detect and quantify them remains particularly important to advancing our understanding of the biology of this disease.
Presently, diagnosis of ALCL is made by tissue biopsy and detection of T-cell markers, such as CD30 and/or CD4. However, mis-diagnosis can occur since some ALCL can be negative for certain markers and/or can be positive for keratin, a marker for carcinoma. Although the ALK-NPM genetic translocation itself can be detected, it is clear that other downstream effectors of ALCL, having diagnostic, predictive, or therapeutic value, remain to be elucidated. Accordingly, identification of downstream signaling molecules and phospho-sites involved in ALK-NPM positive ALCL and development of new reagents to detect and quantify these sites and proteins may lead to improved diagnostic/prognostic markers, as well as novel drug targets, for the detection and treatment of this disease.